DNA polymerases are used for a variety of nucleic acid replication processes in molecular biology, including nucleic acid sequencing, nucleic acid quantification (Real Time PCR, NASBA), and nucleic acid amplification (PCR, RDA, SDA), as well as reverse transcription of RNA into cDNA, nucleic acid labeling, and other processes.
DNA polymerase III holoenzyme (“Pol III”) was first purified and determined to be the principal replicase of the E. coli chromosome by Kornberg (Kornberg, A., 1982 Supplement to DNA Replication, Freeman Publications, San Francisco, pp 122-125, incorporated herein by reference). This holoenzyme is composed of 10 distinct subunits that form three separate functional components (see McHenry, et al., J. Bio Chem., 252:6478-6484 (1977); Maki, et al., J. Biol. Chem., 263:6551-6559 (1988), incorporated herein by reference).
The three components of the Pol III replicase in gram negative bacteria, such as Escherichia coli, are (i) the “core” (i.e. the polymerase), (ii) β (i.e., the sliding clamp), and (iii) the γ-complex (i.e., the clamp loader). In E. coli the τ subunit holds together two cores to form the Pol III′ subassembly, and it binds one γ-complex to form Pol III*. The τ subunit and the γ subunit are both encoded by dnaX. Tau is the full length product, while γ is approximately the N-terminal ⅔ of τ and is formed by a translational frame shift (Tsuchihashi et al., “Translational Frameshifting Generates the γ Subunit of DNA Polymerase III Holoenzyme,” Proc. Natl. Acad. Sci., USA., 87:2516-2520 (1990), incorporated herein by reference). In other gram negative bacteria, such as Aquifex aeolicus, the dnaX gene translates into a single protein (tau) only and the Pol III holoenzyme assembles into the processive replicase without a gamma subunit (Bruck I. et al., “Analysis of a multicomponent thermostable DNA polymerase III replicase from an extreme thermophile.”, J Biol. Chem. 2002 May 10;277(19):17334-48. Epub 2002 Feb. 21, incorporated herein by reference).
Within the “core” are three subunits: the α subunit (encoded by dnaE) represents the catalytical subunit with the DNA polymerase activity; the ε subunit (encoded by dnaQ, mutD) is the proofreading 3′-5′ exonuclease (Scheuermann, et al., Proc. Natl. Acad. Sci. USA, 81:7747-7751 (1984); and DiFrancesco, et al., J. Biol. Chem., 259:5567-5573 (1984), incorporated herein by reference), and the θ subunit (encoded by holE) stimulates ε (Studwell-Vaughan et al., “DNA Polymerase III Accessory Proteins V. theta encoded by holE*,” J. Biol. Chem., 268:11785-11791 (1993), incorporated herein by reference). In E. coli the α subunit forms a tight 1:1 complex with ε (Maki, et al., J. Biol. Chem., 260:12987-12992 (1985) incorporated herein by reference), and 0 forms a 1:1 complex with ε (Studwell-Vaughan et al., supra).
The E. coli Pol III replicase is highly efficient and completely replicates a uniquely primed bacteriophage single-strand DNA (“ssDNA”) genome coated with the ssDNA binding protein (“SSB”), at a speed of at least 500 nucleotides per second at 30° C. without dissociating from a 5 kb circular DNA even once (Fay, et al., J. Biol. Chem., 256:976-983 (1981); O'Donnell, et al., J. Biol. Chem., 260:12884-12889 (1985); and Mok, et al., J. Biol. Chem., 262:16644-16654 (1987), incorporated herein by reference).
DNA polymerase III replicases from a number of gram negative and gram positive bacteria, including thermophilic bacteria, have since been described (for example, see Bullard et al., J. Biol. Chem., 277:13401-13408, 2002; and Bruck et al., J. Biol. Chem., 277:17334-17348, 2002; incorporated herein by reference), and the three-component organization of Pol III replicases in these bacteria appears to be similar to that of E. coli. In Streptococcus pyogenes, for example, the Pol III replicase is comprised of (i) the α subunit encoded by the polC gene (without epsilon and theta subunits, which are missing in gram-positive bacteria), (ii) β-sliding clamp, and (iii) the τ/δ/δ′-complex (i.e., the clamp loader). The assembly of the polC-derived α with β, τ, δ, and δ′ is sufficient to form a functional Pol III replicase in vitro (Bruck I, O'Donnell M., “The DNA replication machine of a gram-positive organism”, J Biol. Chem. 2000 Sep. 15;275(37):28971-83, incorporated herein by reference).
Gram-positive bacteria also contain a second DNA polymerase encoded by the dnaE gene with homology to the E. coli α subunit. Although apparently capable of interaction with β (Bruck and O'Donnell, supra), these dnaE-derived α subunits are much slower than the PolC-derived α subunits of the Pol III replicase, lack proof-reading activity and demonstrate a propensity for interlesional DNA synthesis, suggesting a potential role in induced mutagenesis and DNA repair (Fleft F. et al. “A ‘gram-negative-type’ DNA polymerase III is essential for replication of the linear chromosome of Streptomyces coelicolor A3(2).” Mol Microbiol. 1999 February;31 (3):949-58.), (Le Chatelier E. et al. “Involvement of DnaE, the second replicative DNA polymerase from Bacillus subtilis, in DNA mutagenesis.” J Biol. Chem. 2004 Jan. 16;279(3):1757-67. Epub 2003 Oct. 30.), (Foster K. et al. “DNA polymerase III of Enterococcus faecalis: expression and characterization of recombinant enzymes encoded by the polC and dnaE genes.”, Protein Expr Purif. 2003 January;27(1):90-7.), (Boshoff H I et al. “DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis.”, Cell. 2003 Apr. 18;113(2):183-93.), (Barnes M H et al. “DNA polymerases of low-GC gram-positive eubacteria: identification of the replication-specific enzyme encoded by dnaE.”, J Bacteriol. 2002 July;184(14):3834-8.). In any event, their slow speed and high frame shift frequency (based on the lesion bypass activity) render them unsuitable for use in many molecular biology applications.
The literature has consistently taught that the three principal components of a DNA polymerase III holoenzyme, the core (including the α subunit), the processivity clamp and the clamp loader, are required for a functional DNA replicase having rapid extension rates typical of genomic replication. (See, for example, U.S. Pat. Nos. 6,555,349; 6,221,642; 5,668,004; 5,583,026; 6,677,146; and 6,238,905; see also O'Donnell, Bioessays, 14:105-111, 1992; O'Donnell et al., J. Biol. Chem., 260:12875-12883, 1985; McHenry, Mol. Microbiol., 49:1157-1165, 2003; McHenry, J. Biol. Chem., 266:19127-19130, 1991; Studwell et al., J. Biol. Chem., 265:1171-1178, 1990; Bullard et al., J. Biol. Chem., 277:13401-13408, 2002; and Bruck et al., J. Biol. Chem., 277:17334-17348, 2002. This DNA polymerase Ill literature has clearly established the dogma that the DNA polymerase III α subunit of gram negative bacteria and the polC-encoded α subunit of gram positive bacteria cannot function alone and that other subunits are required for polymerase stability, fidelity, and processivity.
The original reports by Kornberg et al. on the E. coli Pol III were the first to show that the polymerase activity of the α subunit alone or in combination with the core could only be measured at very high enzyme concentrations using a “gap-filling” assay with activated calf-thymus DNA, and produced extension rates for the polymerase core and alpha alone of only 20 b/sec and 7.7 b/sec, respectively. Many more recent reports on Pol IIIs from a variety of bacteria have supported the original finding by Kornberg et al. and further cemented the dogma that the α subunit or the core complex of a DNA polymerase III holoenzyme cannot function in a processive mode with fast extension rates. For example, Bruck et al. (J. Biol. Chem., 275: 28971-28983, 2000) report that the alpha subunit (polC) of Pol III from Streptococcus pyogenes does not function alone as a DNA polymerase, while Bullard et al., J. Biol. Chem., 277:13401-13408, 2002, and Bruck et al., J. Biol. Chem., 277:17334-17348, 2002, report that three components of Pol III are required for replicase activity in Thermus thermophilus and Aquifex aeolicus, respectively.
The low extension rates and high protein concentrations reported in the prior art have prevented the application of bacterial Pol III replicase α subunits alone or in combination with the β sliding clamp to molecular biology research and clinical diagnostics, including the amplification and/or sequencing of nucleic acids.